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 Table of Contents  
Year : 2022  |  Volume : 7  |  Issue : 2  |  Page : 91-96

Seropositivity of COVID-19 asymptomatic nurses using Anti-SARS-CoV-2 nucleocapsid antibodies

1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Najran University, Najran, Saudi Arabia
2 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia

Date of Submission22-Aug-2022
Date of Decision28-Sep-2022
Date of Acceptance02-Oct-2022
Date of Web Publication27-Dec-2022

Correspondence Address:
Dr. Ahmad A Alshehri
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Najran University, Najran 1988
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/KKUJHS.KKUJHS_28_22

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Background: Anti-SARS-CoV-2 antibodies in symptomatic and asymptomatic cases provides helpful insight about its clinical implications. Aims and Objectives: The aim of this study is to determine anti-nucleocapsid IgG antibodies in nurses, qualitatively, both in asymptomatic and symptomatic nurses, and to determine the rate of asymptomatic cases. Second, to compare antibody reactivity from asymptomatic and symptomatic subjects. Materials and Methods: Samples were collected from four hospitals in the Southern Region, Najran, Kingdom of Saudi Arabia (KSA). Quantitative enzyme-linked immunosorbent assay from Epitope Diagnostic, Inc were used to measure the level of anti-nucleocapsid (anti-NC) antibodies in nurses. Results: One hundred twelve samples tested positive for antibodies (70%). Other variables were assessed, including age, gender, ABO blood group, and nationality. A significant difference was found between asymptomatic nurses vs. those with negative antibodies (*P = 0.0147), suggesting they could have transmitted SARS-CoV-2 unknowingly. Forty-one nurses (25.6%) were symptomatic, while 99 nurses were asymptomatic (61.8%). Conclusion: In this study, 112 nurses (70%) tested positive for the anti-NC antibodies, 41 (25.6%) were symptomatic, 99 (61.8%) were asymptomatic, and 48 (30%) were anti-NC antibody negative. Future work should focus on the association of respiratory disease with the concentration of antibodies, and if antibodies wane rapidly after COVID-19 infection.

Keywords: Anti-nucleocapsid antibodies, asymptomatic cases, COVID-19, severe acute respiratory syndrome coronavirus 2

How to cite this article:
Alshehri AA, Hakami AR. Seropositivity of COVID-19 asymptomatic nurses using Anti-SARS-CoV-2 nucleocapsid antibodies. King Khalid Univ J Health Sci 2022;7:91-6

How to cite this URL:
Alshehri AA, Hakami AR. Seropositivity of COVID-19 asymptomatic nurses using Anti-SARS-CoV-2 nucleocapsid antibodies. King Khalid Univ J Health Sci [serial online] 2022 [cited 2023 Jan 30];7:91-6. Available from: https://www.kkujhs.org/text.asp?2022/7/2/91/365758

  Introduction Top

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19.[1] SARS-CoV-2 is transmitted by human-to-human contact through droplets, aerosols, and fomites.[2] Since the outbreak of SARS-CoV-2 in December 2019, the virus is continuing to spread rapidly, with more than 418 million confirmed cases and over 5.8 million global deaths.[3] In addition, the virus continues to evolve with potential new mutant variations.[4] The earliest documented SARS-CoV-2 variants of concern (VOC) were on May 2020 for cases infected with the Beta variant. Other variants were detected during late 2020 including Alpha, Gamma, and Delta on September, October, and November 2020, respectively. The debate continues on how these mentioned variants affect the virus features such as morbidity and mortality rate, diagnostic tools, therapeutics strategies, and public health measures.[5]

COVID-19 patients experience a spectrum of symptoms, ranging from mild to critical. The majority of cases are not severe and patients recover without hospitalization.[6] Asymptomatic infections have been well-documented for COVID-19.[7] A systematic review before the vaccination battle reported that one-third of confirmed COVID-19 cases never experienced symptoms.[8] Nurses have direct contact with patients with SARS-CoV-2, thus increasing exposure to COVID-19. This has raised concern for public health policies, with a risk of asymptomatic transmission.[9]

The serological analysis provides information for surveillance, with an ability to capture subclinical and past infections, which is not achieved by reverse transcription–polymerase chain reaction (PCR).[10],[11] While nucleocapsid (NC) and spike proteins are structural antigens used as biomarkers to detect SARS-CoV-2, the former is highly immunogenic and a serological marker.[12] NC protein elicits a faster immune response than spike protein.[13] Detection of anti-SARS-CoV-2 NC antibodies (anti-NC antibodies) is used as a marker of past COVID-19 infections.

This study aimed to determine anti-NC immunoglobulin G (IgG) antibodies in both asymptomatic and symptomatic nurses and to determine at what extent nurses are asymptomatic. The second objective was to compare antibody reactivity from asymptomatic and symptomatic subjects.

  Materials and Methods Top


A random sampling study was carried out on nurses (N = 160) from four hospitals: King Khalid Hospital, Maternity and Children's Hospital, New Najran Hospital, and Specialized Najran Medical Hospital at Najran city, and Kingdom of Saudi Arabia (KSA). For each subject, we used a structured questionnaire and recorded demographic data: age, gender, nationality [Table 1], and ABO and Rhesus (ABO/Rh) blood grouping. The study was conducted between late December 2020 and mid-January 2021. Participants did not receive COVID-19 vaccines during sample collection.
Table 1: Averages of anti-nucleocapsid antibody levels of all nurses with respective nationality

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Sample collection

For each subject, 5 ml of venous blood was placed in plain tubes to clot at room temperature for 20–30 min, centrifuged at ×4,000 g for 5 min. The resulting serum was collected and aliquoted into 1.5 ml tubes. When testing was not possible on the day of serum preparation, samples were frozen at −20°C until antibody testing could be done. Serum samples with hyperlipemia, hemolysis, and contamination were discarded.

Detection of anti-nucleocapsid antibodies

Anti-SARS-CoV-2 anti-NC antibodies were detected using the EDI COVID-19 NC IgG Quantitative enzyme-linked immunosorbent assay (ELISA) Kit (KT-1034; Epitope Diagnostic, Inc.). Microplate-based immunoassay was used to quantitatively detect anti-NC in sera. Samples were diluted 1:100 by adding 10 μl of serum sample with 1000 μl of COVID-19 IgG sample diluent (#31281 of the kit). Samples were kept at room temperature for 30 min before washing. Between steps, the microtiter plate was washed five times with 200 μl of the ELISA wash concentrate (#10010). The conjugate was an HRP-labeled anti-IgG tracer antibody (#31220), reacting with its substrate (#10020); after 20 min of incubation in the dark, a stop solution was added (#10030). Absorbance was read at 450 nm within 10 min.

Statistical analyses and graphs presentation

GraphPad software (version 8, USA) was used to interpolate samples' optical densities with anti-NC antibodies. Comparisons of the anti-NC Ab concentrations for sex, age, and ABO blood grouping were performed with IBM® SPSS software (Chicago, IL, USA). The analysis of variance test with the nonparametric method was used to statistically analyze symptomatic versus uninfected nurses and those who showed no symptoms with confirmed antibody presence.

Ethical approval

The study was approved by the Najran Health Ethics Committee of the General Directorate of Health Affairs at Najran city, KSA (IRB No. 2020-24E). Written informed consent was obtained from subjects after receiving an explanation of the study.

  Results Top

Standard curve of positive controls and interpolation

The standard curve was plotted using the hyperbola relationship with GraphPad Prism software. Five controls (C1 to C5) were used in duplicate at concentrations of 0, 6.9, 26.3, 100, and 200, respectively. Optical densities of the controls were interpolated in a sigmoidal curve [Figure 1]. According to the manufacturer, the cutoff value was 10 U/mL. Nineteen samples were <10 U/mL (uninfected). Ninety-nine nurses were asymptomatic but positive for the anti-NC antibodies. [Figure 2] shows the standard curve interpolation with the known optical densities for all samples.
Figure 1: Sigmoidal curve of positive controls. Data are presented as means of duplicate samples

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Figure 2: Interpolation of a standard curve with samples' optical densities, showing the concentration of antibodies

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Calculation of cutoff value using negative sera

The cutoff value was calculated using negative sera of absorbance close to background noise, and <0.2 optical density. Having plotted and interpolated the standard curve, the absorbance of negative sera calculated the cutoff value by taking the mean of negative sera at ± 2 standard deviations [Figure 3]. One hundred twelve samples were positive, from 160 with this calculation as the cutoff, and 48 with negative results. Likewise, 48 samples were negative after interpolating standard curve readings [Figure 2].
Figure 3: Calculation of the cutoff value using negative sera ± SD. SD: Standard deviation

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Symptomatic versus asymptomatic nurses

The difference between symptomatic nurses confirmed to have COVID-19 and those who had not been infected, or was asymptomatic [Figure 4], was statistically significant (****P < 0.0001; *P = 0.0147). This suggests that anti-NC antibodies were specific and could predict symptomatic cases of SARS-CoV-2 infections. Another observation was that the asymptomatic cases differed statistically from uninfected subjects. [Figure 4] shows that there were 41 symptomatic nurses and 99 asymptomatic nurses.
Figure 4: Symptomatic versus asymptomatic and uninfected nurses. Statistical analyses were calculated using one-way ANOVA with the nonparametric method. ****P < 0.0001. ANOVA: Analysis of variance

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Antibody level versus time of infection

To trace antibody concentration after months of COVID-19 infection, nurses were asked through a questionnaire about the time they had been confirmed by PCR: of 41 symptomatic nurses, data on infection time were recorded for 37 subjects. Optical densities were plotted in the time in which a serum sample was collected for anti-NC antibody testing. In most, serum samples were collected less than 300 days from infection. One sample was drawn from a nurse after 324 days of confirmed COVID-19 diagnosis. It was noted that a statistically significant inverse correlation was found [Figure 5].
Figure 5: Duration (days) versus optical densities of the symptomatic nurses

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Comparisons with age, gender, ABO blood grouping, and nationality

Using the interpolated concentrations of the anti-NC Ab, comparisons with sex, age, ABO blood grouping, and nationality were performed. Females represented the highest percentage with elevated antibody concentration. However, the male gender represented around 18% of the samples. Regarding ABO/Rh, the blood groups O+, B+, and A + were linked with the highest concentration of the anti-NC Ab, respectively, compared to other blood groups. However, the sample size was too small to be included in statistical analyses, and no significance was seen between O+, B+, and A + in terms of the anti-NC antibody concentration. With regard to nationality, 74 were Indians, 33 were Filipinos, and 32 were Saudi Arabians. While these three nationalities dominate, Filipino nurses showed the highest median value of anti-NC antibodies, followed by Indians and Saudi Arabian nurses [Figure 6].
Figure 6: Comparison of antibodies with age, gender, ABO/Rh blood groups, and nationality. ABO/Rh: ABO and Rhesus

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  Discussion Top

This article focuses on the seropositivity of SARS-CoV-2 asymptomatic nurses using anti-NC antibodies. Anti-NC antibody concentration of positive controls was measured from the standard curve [Figure 1]. After determining control optical densities, absorbances of nurses' sera that reacted to detecting anti-NC antibodies were interpolated with controls [Figure 2]. The cutoff value was calculated from negative sera [Figure 3]. Negative and positive samples were the same after using the two methods in [Figure 2] and [Figure 3]: 48 negative sera and 112 positive sera. We observed 99 subjects as asymptomatic and 40 as symptomatic [Figure 4], but anti-NC antibodies were negative in 48 of 160 nurses.

Serological testing to understand the kinetics of anti-SARS-CoV-2 antibodies is rapidly evolving. The kit used in this study can successfully differentiate between the uninfected population from positive patients. Pilmis et al. was a similar study to our own. Of 3,062 health-care workers, the serological status of anti-NC was positive in 8.4%, with age correlation using the anti-NC index.[14] In a study to evaluate antibodies that were declining in Northern Italy, anti-NC IgG antibodies started to wane after 4 months, but they found that anti-spike (anti-S) antibodies were inversely correlated with anti-NC. Around 78% (n = 1159) of the seronegative anti-NC individuals were positive for anti-S IgG antibodies.[15] In a more detailed monitoring study, anti-NC and anti-S antibodies decreased after 38 weeks of infection, as some patients turned seronegative-but after infection, positivity could be detected in 90% of individuals after 9 months.[16] A Malaysian study using 148 healthcare employees confirmed that anti-NC antibodies wane faster than anti-S antibodies.[17] A cohort study by Beretta et al. claimed that the majority of SARS-CoV-2-infected patients tested positive for anti-NC antibodies after 12 months of symptoms.[18] They used a qualitative rapid test, developed and used extensively during the COVID-19 pandemic. Rapid detection assays are fast but limited by low sensitivity. ELISA tests provide more sensitive assays of antibodies.[13],[19],[20],[21]

Our study incorporated other variables, namely: age, gender, ABO blood grouping, and nationality. In relation to the ABO blood group, Gil-Manso et al. showed that non-O group individuals required a longer period to clear SARS-CoV-2.[22] Regarding race, it was noticeable that the level of anti-NC antibodies was slightly higher among Filipino nurses, although their number was less than Indian nurses. This difference is not statistically significant [Figure 6]. Despite that gender differences in SARS-CoV-2 pathogenesis are poorly understood, female participants showed less antibodies.[16] However, akin to age and ABO blood grouping, it is challenging to draw a definitive conclusion from our observation.

One study limitation is that the vaccine targets the spike protein, while our study focused on antibodies against the NC. Moreover, only three nationalities dominated the study compared to others. Male nurses represented around 18% of the study, which might have impacted the correlation regarding gender. In this study, we attempted to use as strong samples as possible. However, the two kits in this study were somewhat expensive, which was one of the limitations to increase the sample size with replicates. We concluded that 112 nurses (70%) tested positive for anti-NC antibodies, 41 (25.6%) were symptomatic, and 99 (61.8%) were asymptomatic. Forty-eight subjects (30%) were anti-NC antibody negative.

  Conclusion Top

This study has been conducted before the approval of the COVID-19 vaccine in Saudi Arabia; therefore, it applies to strains circulating then. However, other SARS-CoV-2 VOCs have been emerged; Saudi Arabia has not reported any distribution of documented VOC at the time of study conduction.[23] Effective antivirals are needed to combat these variants, and several articles have identified potential inhibitors.[24],[25],[26] Future work should include more detailed investigations, e.g., the association of respiratory symptoms with immunological assay results, anti-SARS-CoV-2 antibodies over time following the initial confirmed COVID-19 infection, and the association with the detected virus variant.

Author contributions

A. A. A.: Study design, funding acquisition, methodology, data collection, data interpretation, writing original draft and approval of the final manuscript to be published, A. R. H.; data interpretation, statistical analyses, graphs presentation, writing original draft, editing, and approval of the final manuscript to be published.

Data availability statement

All study data are included in the article.

Financial support and sponsorship

This research was funded by Scientific Research Deanship at Najran University, KSA (No. NU/MID/18/029).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1]


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